Abstract
Single-cell barcoding enables the combined processing and acquisition of multiple individual samples as one. This maximizes assay efficiency and eliminates technical variability in both sample preparation and analysis. Remaining challenges are the barcoding of live, unprocessed cells to increase downstream assay performance combined with the flexibility of the approach towards a broad range of cell types. To that end, we developed a novel antibody-based platform that allows the robust barcoding of live human cells for mass cytometry (CyTOF). By targeting both the MHC class I complex (beta-2-microglobulin) and a broadly expressed sodium-potassium ATPase-subunit (CD298) with platinum-conjugated antibodies, human immune cells, stem cells as well as tumor cells could be multiplexed in the same single-cell assay. In addition, we present a novel palladium-based covalent viability reagent compatible with this barcoding strategy. Altogether, this platform enables mass cytometry-based, live-cell barcoding across a multitude of human sample types and provides a scheme for multiplexed barcoding of human single-cell assays in general.
Highlights
First mass cytometry-specific barcoding approaches have relied on labeling cells with heavy-metals via amineor sulfhydryl-reactive chelating agents[16,17]
We conjugated antibodies against beta-2-microglobulin (b2m) as part of the MHC class I complex as well as antibodies against the beta-3 subunit of the Na+/K+-ATPase (CD298) to heavy-metal isotopes for their use in mass cytometry (Fig. 1A). We tested their expression on various cell populations, including immune cell subsets found in whole blood (Fig. 1B,C, see Table S1), as well as various cancer and non-immune cell lines such as leukemic (U937, Ramos, HEL, Jurkat, REH and THP-1), embryonic or stem cell-derived (293 T, H9 human embryonic stem cells and NTERA) and carcinoma cell lines (A549, NCI-H460, HCT 116 and HeLa; Fig. 1D)
Cellular barcoding approaches have been proposed for a variety of technologies, including single-cell sequencing[30], antibody/sequencing hybrid technologies[31], genetic barcoding for cell lineage tracing[32], fluorescent barcoding for flow cytometry[33] and more recently heavy-metal isotope based barcoding in mass cytometry[17,18,19,34]
Summary
First mass cytometry-specific barcoding approaches have relied on labeling cells with heavy-metals via amineor sulfhydryl-reactive chelating agents[16,17]. As these groups are most abundantly found within cells, as opposed to their surface, fixation and permeabilization are required, making these methods less suitable for barcoding before probing of fixation- or permeabilization-sensitive molecules or epitopes. These issues can be overcome by making use of cell-surface molecules for barcoding purposes. We demonstrate broad applicability of this approach in research involving human stem cells, immune cells as well as a broad range of different cancer cell lines and patient samples
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